SUMMARY This new R01 builds on discoveries during the R37 period (2008-2018) that established epigenetic mechanisms (miRNAs, histone modifications) regulating osteoblast differentiation. We characterized for the first time a ?signature? of specific histone modifications that are associated with dynamic changes in gene expression during the temporal progression of osteogenesis. These histone modifications also predicted ?enhancers?, which are critical cis-regulatory elements that contribute to local gene expression. We now propose to examine the recently recognized ?super enhancer? domains (SEDs) that include regulatory elements for multiple transcription factors that have emerged as key regulators of cell phenotypes. SEDs function in chromatin organization via long range intra- and inter-chromosomal interactions that coordinate control of gene cohorts responsible for lineage specification and distinct cell identity. Our preliminary studies have identified a subset of SEDs that we now propose are putative ?bone-essential super-enhancers? and candidates for the important decision stage of commitment to osteogenesis from MSCs. We hypothesize that super-enhancer domains are differentially activated from the undifferentiated MSC to the osteoblast commitment stage, and function to establish the osteogenic phenotype by coordinately regulating gene networks and contributing to higher order chromatin organization that supports cell identity. Our studies will in: Aim1- analyze the functional effects of prioritized SEDs we have identified related to osteoblastogenesis and mature bone activities through directed inhibition and activation of SEDs using CRISPR/Cas9 in MSCs; Aim 2- determine the chromosomal domains that interact with SEDs to control multiple genes and networks that commit MSCs to the osteoblast phenotype through chromatin organization; and Aim 3- demonstrate in mouse models that using CRISPR activated SEDs in MSCs will stimulate bone formation. Impact: These studies pioneer a new level of gene regulation for MSC lineage commitment to osteogenesis, based on an emerging understanding of SED functions in other tissues but have been minimally studied in bone. By characterizing SED mechanisms related to chromatin organization and stabilization in MSCs, we will discover novel mechanisms of multi-dimensional coordinate control of transcriptional hubs and protein complexes within an SED that is responsible for establishing commitment to the osteoblast phenotype. Importantly, knowledge of the chromatin organization that stabilizes the osteogenic phenotype impacts on future novel treatment strategies for skeletal disorders.